This invention relates to a system for investigating particles, and in particular to such a system wherein sorted particles are selected at a yield/purity ratio at very high signal processing rates.
Systems for sorting minute particles in a fluid may be referred to as flow cytometer sorting systems. These systems are used in the medical research and diagnostic fields for the rapid analysis and processing of biological cells. Systems for sorting particles suspended in a liquid according to certain characteristics are discussed in U.S. Pat. No. 3,963,606, 4,063,284 and 4,487,320. Specifically, in these prior art systems optical measurements of characteristics of each particle of a group of particles are made while the particles are suspended in a liquid. In one such system, a flow cytometer cell sorter analyzes cells or particles in suspension which are labeled with a fluorescent marker and are carried single file in a fast-moving liquid stream sequentially through a tightly focused laser beam whose wavelength is adjusted to illuminate the fluorescent dye (Bonner, W. A., Hulett, H. R., Sweet, R. G., and Herzenberg, L. A., Fluorescence Activated Cell Sorting, Rev. Sci. Instrum. 43:3 404, 1972). The fluorescence produced during the laser beam crossing is collected by an optical system and projected through spectral filters onto photodetectors. These detectors convert the fluorescence to electrical pulses whose amplitudes depend on the total light reaching the detectors. Particles having predetermined characteristics are recognized by their distinctive fluorescence. After measurements of the characteristics, each particle flows in a liquid jet stream until the particle reaches a point where the jet breaks into discrete droplets. At this point, an electric charge is induced on each droplet containing a particle to be sorted and the charged and uncharged droplets are then sorted electrostatically into appropriate recovery vessels.
Previously proposed methods of separating particles have also included separation on the basis of electronically measured cell volume by charging cells according to a sensed volume and detecting the charged cells in an electrostatic field for separation into collection vessels. (Fulwyler, M. J., Electronic Separation of Biological Cells by Volume, Science 150:910, 1965). A system has also been proposed for quantitative analysis and sorting of particles based on multiple parameters to increase the ability to differentiate among cell types by measuring the differences between multiple parameters of the cells (Steinkamp, et al., A New Multiparameter Separator for Microscopic Particles and Biological Cells, Rev. Sci. Instrum., Vol. 44, No. 9, 1301 (1973). In this system, cells stained with fluorescent dyes in liquid suspension enter a flow chamber where electrical and optical sensors measure cell volume, single and two color fluorescence and light scatter to produce electrical signals. A processing signal unit processes the electrical signals according to preselected parameters and electrically charges droplets containing the desired cells. The typical rate at which cells are sorted in this system is a few hundred cells/sec.
A number of proposals have been described for multiple parameters, increasing processing speeds and accuracy of flow cytometer cell sorters. For example, systems have been proposed for electronics modularization to process as many as eight input parameters for sorting cells (Hiebert, R. D., Jett, J. H., and Salzman, G. C., Modular Electronics for Flow Cytometry and Sorting: the Lacel System, Cytometry, 1:337, 1980); a system operating at rates of 15,000-25,000 cells/sec by increasing the pressure in the jet stream to increase droplet production frequency (Peters, D., Branscomb, E., Dean, P., Merrill, R., Pinkel, D., Van Dilla M., and Gray, J. W., The LLNL High-Speed Sorter: Design Features, Operational Characteristics, and Biological Utility. Cytometry 6:290, 1985; Peters, D., Dean, P., and Merrill, J. T., Multi-Parameter, Computer Controlled Operation of a FACS II Cell Sorter, Cytometry 2:350, 1982). Examples of systems for increasing accuracy are: a system to assure proper timing (Martin, J. C., McLaughlin, S. R., and Hiebert, R. D., A Real Time Delay Monitor for Flow-System Cell Sorters, J. Histochem. and Cytochem., 27(1):277, 1979), sort timing a system for increasing droplet breakoff stability (Stovel, R. T., The Influence of Particles on Jet Breakoff, J. Histochem. and Cytochem. 25(7):813, 1977; Auer, R. E., Method and Apparatus for Detecting Change in the Breakoff Point in a Droplet Generation System (analysis of blood cells), U.S. Pat. No. 4,487,320, 1984; Auer, R. E., Method and Apparatus for Detecting Change in the Breakoff Point in a Droplet Generation System (radiation sensing means), U.S. Pat. No. 4,691,829, 1987); a system describing individual cell sorting (Stovel, R. T., Individual Cell Sorting, J. Histochem. and Cytochem. 27(1):284, 1979); a system correlating multiparameter data for each cell (Parson, J. D., Hiebert, R. D., and Martin, J. C., Active Analog Pipeline Delays for High Signal Rates in Multistation Flow Cytometers, Cytometry 64:388, 1985); and a system for parallel processing a signal from a large number of detectors (van den Engh, G., and Stokdijk, W., Parallel Processing Data Acquisition System for Multilaser Flow Cytometry and Cell Sorting, Cytometry 10:282, 1989). Further, systems for improving the processing of electronics of flow cytometry applications with computers have been proposed (Steinkamp, J. A., Fulwyler, M. J., Coutler, J. R., Hiebert, R. D., Horney, J. L., and Mullaney, P. F., A New Multiparameter Separator for Microscopic Particles and Biological Cells, Rev. Sci. Instrum. 44:1301, 1973; Steinkamp, J. A., and Hiebert, R. D., Signal Processing Electronics for Multiple Electronic and Optical Measurements on Cells, Cytometry 2(4):232, 1982), and computer systems for automating cells systems with multiple samples based on multiple parameters (Arndt-Jovin, D., and Jovin, T. M., Computer-Controlled Multiparameter Analysis and Sorting of Cells and Particles, J. Histochem. Cytochem. 22:622, 1974; Arndt-Jovin, D., and Jovin, T. M., Automated Cell Sorting With Flow Systems, Ann. Rev. Biophys. Bioeng. 7:527, 1978). The above described proposals having the advantage of more accurately sorting cells have not allowed for flexibly determining sorting decisions based on the particular experiment performed.
In the experiments performed when objects (e.g. cells) are separated, i.e. sorted by a flow cytometer/cell sorter, at relatively slow event rates the individual objects are usually spaced a distance from each other which is sufficient to allow the objects to be separated individually. However, when the objects are sorted at higher rates, objects may be closer together and multiple objects may be present within a single sorting unit, i.e. "droplet". This condition of having multiple objects within a single sorting unit is considered to be an "overlap" or "coincident" condition. Further, when objects are coincident, some fraction of the sorted objects are undesired resulting in a "contamination" of the sample.
The yield of the system is described as the number of events which are sorted by the system. Purity of the sample is described as the percentage of the sample which does not contain undesired objects sorted due to coincidence with desired objects. When a coincident condition occurs, if all the objects which are coincident are of the same type ("friends"), i.e. all the objects will be sorted into the same recovery vessel, then no losses of yield or purity will be observed. If, however, the objects which are coincident are of different types ("foes") the objects which are sorted will have a loss of purity unless the coincident objects are sorted from the desired objects. In the previously described systems, the additional sorting of coincident objects from objects of interest would lead to a loss of yield.
In the previously proposed systems which make a determination of a coincident condition, the systems take one of two approaches in determining the sorting of coincident events: either the systems do not sort any particles which are determined to be coincident or the systems ignore the coincident condition and sort all particles whether or not the particles are coincident (Steinkamp, J. A., and Hiebert, R. D., Signal Processing Electronics for Multiple Electronic and Optical Measurements on Cells, Cytometry 2(4):232, 1982). There is no consideration taken as to whether particles are "friends" or "foes". The first approach results in a sample having all contamination removed from the sample. In this approach a condition of "maximum purity" is achieved and all sorting units with contaminating particles are discarded. In this case, the contaminating particles may be either "friends" or "fores". If they are "friends" and discarded, an unnecessary loss of yield is realized. The second approach results in a sample having a contamination of the sample with particles which are undesired, and therefore, results in a sample having less than "maximum purity" . The second approach may be used in experiments in which the purpose of the experiment is to recover as many particular objects as possible (i.e. "maximum yield"). Moreover, in this type of experiment contamination is not important to the user and any sorting unit containing desired objects is recovered whether or not the sorting unit also contains a contamination.
A proposal has been made for sorting decisions with droplet charging control electronic circuitry to handle independent input signals for sorting cells in sort directions based on a sorting decision scheme employing a variety of coincidence and anticoincidence requirements (McCutcheon, M. J., and Miller, R. G., Flexible Sorting Decision and Droplet Charging Control Electronic Circuitry for Flow Cytometer-Cell Sorters, Cytometry 2:219, 1982). In this system, a Read Only Memory (ROM) stores 16 separate sorting programs for making sorting decisions based on multiple parameters. The sorting decisions are inputted to circuits which exclude invalid sorting coincidences for cells which are detected separately yet close together in time as to result in conflicting sorting requirements. The system provides for three levels of control over sorting conflicts: 1) treat as unimportant the presence of contamination from other sorting fractions in a given sorting fraction; 2) recognize that a particular class of cells is undesirable and have the system lock out valid sorting events occurring after the undesirable cell; and 3) prevent contamination of the sorted cell by events still being processed. In that system, all events which are considered coincident are either aborted or ignored with no regard to their "friend" or "foe" status. Therefore, the system only allows sorting at either maximum purity or maximum yield.
This invention makes use of a recognition that it is desirable for particular experiments to have a contamination level which is settable by the user. Therefore, the present system sorts particles at a selected yield/purity ratio which ratio can include an intermediate value of the maximum yield and maximum purity. Moreover, the present invention flexibly sorts particles based on the needs of each individual experiment or assay, unlike prior art systems which fix sorting of particles based on either maximum purity or maximum yield. Therefore, a particular experiment which might be able to tolerate a contamination rate, for example 10% contamination, would be able to achieve a higher yield of sorted particles than if the same experiment was forced to use a system based on maximum purity. Also, unlike prior art systems, the present invention takes into consideration "friend" or "foe" status of neighboring events when determining coincidence for a more precise control of purity/yield considerations.
An additional advantage of the present system over prior art systems is that the quality of experiments at both high and low speeds can be improved. The quality of experiments at any speed can be affected by the random arrival nature of events within a sorting unit in some systems (e.g. flow cytometer/cell sorter). The sorting unit, whether defined by droplet, fluidic switching, zapping or other technology, can be considered to be a queuing length. Moreover, the arrival statistics of particles to this queue can be random or non-random. Random arrival can be described by queuing theory using poisson distribution functions. Other mathematical models may describe non-random arrival statistics of particles to the queue. Predictions of particle arrival and servicing (e.g. serial or parallel processing) in queues, including multiple path and/or multiple queues can also be described. This invention makes use of the arrival statistics and servicing requirements in the queue to separate particles on the basis of yield or purity or any desired combination or ratio of these quantities.
In an exemplary and nonlimiting embodiment of the invention, a system trigger accepts incoming analog input signals, for example from commercially available particle detectors. The input signals are produced by the particle detector for all cells which pass through the system. The input signals may be generated when the surface of a cell passes by a selected point of the particle detector, i.e. light scatter in a flow cytometer/cell sorter. In operation of the system, an input signal above a user determined threshold value triggers the initiation of the system and synchronizes parts of the system such as the event timer, inter-event timer and traditional data acquisition sort system.
In this embodiment of the system, an event timer is provided to measure the event time from when an input signal triggers the system to the time at which a sort pulse should be applied to a particle, i.e. a charge pulse should be applied near droplet breakoff of a flow cytometer for droplet sorting. The event time is a relative constant value that is defined by the physical separation of the signal excitation source from the location at which the sorting will occur and the velocity at which the object is traveling between these points. The event timer provides a signal to the system at the time a sort pulse should be applied to a particle.
This implementation includes an inter-event timer responsive to successive trigger signals to measure the time between system triggers. The time between system triggers is used to measure the distance between the cells and to determine if the distance is less than a user determined selected interval. If the distance is less than the selected interval, the measured cells are considered to be overlapping or coincident. The system stores the inter-event timer measurements in a buffer for use with sorting logic circuitry in making a sort decision.
In this embodiment, the sorting logic circuitry is responsive to the trigger signal, the inter-event timer measurements and respective detection signals which correspond to selected parameters of the particles to be sorted. The sorting logic circuitry operates on a user-selectable function of the inter-event timer measurements and the respective detection signals, hereafter called the sort decision logic condition. The sort decision logic condition can be selected by the user to correspond to a yield/purity ratio. In response to the sort decision logic condition, meaning that the object was of interest, a sort pulse is generated. After receiving an event timer signal, the sort pulse is applied to the object of interest and the object is sorted to an appropriate container.